Nucleic acid drugs have been attracting attention as drugs of next generation, but in order to efficiently introduce nucleic acids into cells, a carrier is required. The carrier is roughly divided into a virus carrier and a non-virus carrier. The virus carrier has a high introduction efficiency of nucleic acid in comparison with the non-virus carrier, but cannot solve the problem of toxicity. Therefore, the non-virus carrier has been attracting attention because it has high safety although the introduction efficiency is inferior to the virus carrier.
As the non-virus carrier, a carrier using a lipid membrane composed of a cationic lipid, a PEG lipid or the like and a carrier using a micelle composed of a cationic polymer have been reported, and investigations on the carrier using a lipid membrane which can control the function by the composition of the lipid have been actively made. Of the lipids for use in the lipid membrane, the lipid which most affects the introduction efficiency of nucleic acid is a cationic lipid.
The cationic lipid ordinarily has one quaternary ammonium group and two hydrophobic groups. Typical cationic lipid includes N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (hereinafter DOTAP-Cl) and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (hereinafter DOTMA-Cl). These lipids are the cationic lipids most widely used because of the high introduction efficiency of nucleic acid in comparison with other known cationic lipids.
DOTAP-Cl and DOTMA-Cl are obtained by introducing an oleoyl group or an oleyl group into dimethylaminopropanediol and then subjecting to quaternization with methyl chloride (Non-Patent Document 1). However, since methyl chloride is gas under normal temperature and atmospheric pressure, it must be used a high-pressure reaction vessel, for example, an autoclave. In addition, since methyl chloride is highly virulent, there is a drawback in that particular attention to safety must be paid to its handling and a device for use.
For the reason described above, a method for producing DOTAP-Cl or DOTMA-Cl without using methyl chloride has been developed and reported. In Non-Patent Document 2, an oleoyl group is introduced into dimethylaminopropanediol, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium iodide (hereinafter DOTAP-I) is synthesized by using methyl iodide, and then the iodide ion of DOTAP-I is exchanged with a chloride ion by a column packed with an ion exchange resin.
However, the method described in Non-Patent Document 2 requires a large amount of ion exchange resin to the lipid obtained. Moreover, in order to perform efficient anion exchange, since the concentration of DOTAP-I in the solution must be diluted, it is necessary to dissolve DOTAP-I in a large amount of the solvent so that the production scale becomes very large. In addition, since a large amount of the solvent must be removed after the anion exchange in the ion exchange column, the method is inefficient and not preferred industrially.
Therefore, in Patent Document 1, anion exchange is carried out by repeating four times the operation of adding a methanol solution of 1 N hydrochloric acid to a dichloromethane solution of N-[1-(2,3-dilinoleoyloxy)propyl]-N,N,N-trimethylammonium iodide or N-[1-(2,3-dilinoleyloxy)propyl]-N,N,N-trimethylammonium iodide, stirring and washing with a sodium chloride solution. However, this method not only has a low anion exchange ratio but also requires an inefficient purification step, for example, column purification, in order to obtain a product having the purity required for pharmaceutical, because in the case of having an ester bond in the molecule as in DOTAP-Cl, an impurity, for example, a fatty acid is generated by hydrolysis with an acid.